The overall goal of this project is to develop a safe and broadly effective vaccine that will prevent group A streptococcal infections. Previous studies have shown that the,surface M proteins, which are the major protective antigens, contain tissue-crossreactive epitopes as well as protective epitopes. The serotype- specific protective epitopes may be separatedfrom potentially harmful autoimmune epitopes by using limited N-terminal peptides of M proteins. The protective M protein fragments representing multiple serotypes of group A streptococci may then be combined to form a multivalent vaccine. The specific aims of this proposal are: 1) To identify the primary structures of M proteins or other surface proteins that contain opsonic (protective) epitopes from serotypes of group A streptococcithat are epidemiologically important and, therefore, necessary vaccine components, 2) To construct recombinant, multivalent vaccines that evoke optimal opsonic antibody responses in laboratory animals against 26 different serotypes of group A streptococci, 3) To test immune rabbit sera evoked by multivalent vaccines for opsonic and bactericidal antibodies against clinical isolates of group A streptococci collected from children with pharyngitis in 10 geographic sites in the U.S., 4) To develop strategies of intranasal delivery of multivalent Mprotein-based vaccines that result in secretory and systemic immune responses, and 5) To directly compare the protective immunogenicity of multivalent M protein-based vaccines delivered to mice via either the intramuscular or intranasal routes. In studies accomplished during the previous period of funding, we made considerable progress in achieving our aims. The 26-valent vaccine has now completed phase 2 clinical trials in adults and will soon be tested in children as an injectable vaccine. Our proposed studies will focus on optimal formulation of multivalent vaccines designed for mucosal (intranasal) delivery. Because mucosal delivery of streptococcal vaccines may have both immunological and practical advantages over parenteral delivery, we will assess different strategies of intranasal delivery and then directly compare the protective efficacy of i.n. vs i.m. vaccines in mice. In addition, we will continue our studies to assess the potential clinical efficacy of the 26-valent vaccine using in vitro opsonization assays and clinical isolates of group A streptococci representing vaccine and non-vaccine serotypes collected in our ongoing epidemiologic studies. New projects will focus on the function of the N-terminal regions of M proteins, the results of which we believe will add significantly to our understanding of the biology of these organisms and potential avenues for improving the efficacy of new vaccines. Overall, these studies should provide the detailed information needed to develop a safe and effective multivalent vaccine that could prevent the majority of streptococcal infections in North America and Western Europe. [unreadable]